Cross-linked hyaluronic acid and collagen and uses thereof

ABSTRACT

The present invention discloses a cross-linked hyaluronic acid/collagen formulation which has improved composition for dermal filling and higher persistence than cross-linked collagen or HA alone. Also disclosed are methods for preparing cross-linked hyaluronic acid/collagen formulations and using such for augmenting soft tissues in mammals.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/247,175, filed on Oct. 7, 2008, which claims priority to U.S.Provisional Patent Application Ser. No. 60/978,423 filed on Oct. 9,2007, the entire disclosure of each of these applications beingincorporated here by this reference.

FIELD OF THE INVENTION

The present invention relates in general to compositions and methods forproducing materials for soft tissue augmentation. More specifically, theinvention provides a cross-linked hyaluronic acid (HA) and collagen(Cgn) implant of improved composition and stability (“persistence”) foraugmenting soft tissue in mammals.

BACKGROUND OF THE INVENTION

Both collagen and HA are naturally found in the skin. Although thecollagen:HA ratios vary with age, sun exposure, type of skin, and otherfactors, matching the skin's composition could have a positive effect onduration and biocompatibility of a dermal filler.

SUMMARY OF THE INVENTION

The present invention relates to cross-linked HA and collagen implantsfor augmenting soft tissue in mammals. In one aspect, the inventionfeatures a method for preparing an implant. The method comprisescontacting HA with a cross-linker to allow cross-linking of the HA bythe cross-linker, thereby forming a first composition; contacting thefirst composition with collagen to allow cross-linking of the collagenby the cross-linker, thereby forming a second composition; andcontacting the second composition with a HA solution to allowcross-linking of the HA in the solution by the cross-linker, therebyproducing an implant.

Many cross-linkers may be used in a method of the invention, includingand not limited to divinyl sulfone (VS), 1,4-butanediol diglycidyl ether(BDDE), ultraviolet radiation, gamma radiation, electron beam radiation,and glutaraldehyde. For example, when VS is used as the cross-linker,the concentration of the VS for forming the first composition may beabout 500-10,000 ppm, and preferably about 5000 ppm. The cross-linkingof HA with VS for forming the first composition may occur at about50-60° C.

When practicing the method of the invention, the concentration and thepH of the HA for forming the first composition may be about 30 mg/ml orhigher and about 9-12, respectively. In some embodiments, the collagenfor contacting the first composition is soluble; in some embodiments,the concentration of the collagen for contacting the first compositionis about 10-50 mg/ml, preferably about 30 mg/ml; in some embodiments,the pH of the collagen for contacting the first composition is about2-3; in some embodiments, the collagen for contacting the firstcomposition is type I, II, III, IV, or V collagen, or a combinationthereof. In some embodiments, the pH of the second composition is about6-8, and preferably about 7. The concentration of the HA solution forcontacting the second composition may be about 3-15% weight/volume, andpreferably about 5-10% weight/volume. The cross-linking of the HA in thesolution by the cross-linker may occur at a pH of about 7.0-7.6.

A method of the invention may further comprise contacting the secondcomposition with a local anesthetic agent such as lidocaine. Theconcentration of the lidocaine may be about 1-7 mg/ml, and preferablyabout 2.7-3.3 mg/ml, in the implant.

An implant prepared according to a method described above is within theinvention. In particular, the present invention provides an implantcomprising a core of HA cross-linked by a cross-linker, a middle layerof collagen surrounding and cross-linked by the cross-linker to thecore, and an outer layer of HA surrounding and cross-linked by thecross-linker to the collagen layer. As mentioned above, the cross-linkermay be VS, BDDE, ultraviolet radiation, gamma radiation, electron beamradiation, or glutaraldehyde. The implant may further comprise about 10ppm or less of cross-linker not cross-linked to the HA or collagen. Insome embodiments, about 5-50% of the amine groups on the collagen aremodified by the cross-linker. In some embodiments, the elastic modulusof the implant is higher than about 200 Pascals or the implant may havean extrusion force through a 24 or larger gauge needle of about 50Newtons or less.

The invention is also directed to an implant prepared by the methoddescribed above, wherein the crosslinker is VS or BDDE, wherein theconcentration of the VS for forming the first composition is about500-10,000 ppm, preferably about 5000 ppm, and wherein the cross-linkingof the HA by the VS for forming the first composition occurs at about50-60° C. In some embodiments of the implant prepared by the methoddescribed above, the concentration of the HA for forming the firstcomposition is about 30 mg/ml or higher and the pH of the HA for formingthe first composition is about 9-12. In some embodiments of the implantprepared by the method described above, the collagen for contacting thefirst composition is soluble and the concentration of the collagen forcontacting the first composition is about 10-50 mg/ml, preferably about30 mg/ml. In some embodiments of the implant prepared by the methoddescribed above, the pH of the collagen for contacting the firstcomposition is about 2-3 and the collagen for contacting the firstcomposition is type I, II, III, IV, or V collagen, or a combinationthereof. In some embodiments of the implant prepared by the methoddescribed above, the pH of the second composition is about 6-8,preferably about 7. In some embodiments of the implant prepared by themethod described above, the concentration of the HA solution forcontacting the second composition is about 3-15% weight/volume,preferably about 5-10% weight/volume. In some embodiments of the implantprepared by the method described above, the cross-linking of the HA inthe solution by the cross-linker occurs at a pH of about 7.0-7.6.

The present invention also features a packaged product. The productcomprises a syringe and a needle. The syringe is loaded with an implantof the invention.

The invention further provides a method for filling voids and defectsand increasing tissue volume in a mammal. The method comprisesadministering to a mammal an implant of the invention. The implant maybe administered by intradermal or subcutaneous injection.

The above mentioned and other features of this invention and the mannerof obtaining and using them will become more apparent, and will be bestunderstood, by reference to the following description, taken inconjunction with the accompanying drawings. The drawings depict onlytypical embodiments of the invention and do not therefore limit itsscope.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart illustrating a representative process for HAVSCgnpreparation.

FIG. 2 shows elastic and viscous modulus during a frequency sweep ofHAVSCgn.

FIG. 3 depicts effects of hyaluronidase (enzymatic) degradation ofHAVSCgn relative to Captique™ on the elastic modulus.

FIGS. 4A and 4B are FT-IR (fourier transform—infrared) traces foruncross-linked HACgn and cross-linked HAVSCgn, respectively.

FIG. 5 shows persistence of HAVSCgn relative to Captique™ andCosmoPlast®.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the unexpecteddiscovery that HA cross-linked at a high concentration and then coatedwith collagen and HA produces a long lasting implant. More specifically,the invention relates to HA and collagen cross-linked using VS toformulate a physiologically-matched wrinkle corrector, which is referredto as HAVSCgn.

In general, HA is first cross-linked at a high concentration. Theresidual cross-linker is then reacted with collagen followed by theaddition of a HA solution for complete utilization of the cross-linkerand a laminating effect. The resulting gel has a high elastic modulusyet can still be extruded through a fine gauge needle with minimalforce. The HA and collagen combination mimics the natural components ofthe skin and is an ideal composition for augmenting soft tissue inmammals. The HA and collagen gel of the present invention has improvedrheological properties and volume stability or persistence compared to asingle component formulation of HA, such as Captique™, or a singlecomponent formulation of collagen, such as CosmoPlast®.

The process of the present invention involves obtaining HA either insolid or liquid form and cross-linking HA as a concentrated solution,followed by the addition of collagen and finally more HA. The collagenof the present invention primarily derives from mammalian sourcematerials, such as bovine or porcine corium or tendon or human placentaltissue. Collagen produced from human fibroblast cell culture, orrecombinantly-produced collagen expressed from a cell line may also beused.

Base (e.g., NaOH) is first added to HA to adjust the pH to 9-12. Across-linker is added to the HA and the reaction is allowed to proceed.Collagen (pH 2-3) is then added. The collagen reacts with thecross-linker to covalently bind to HA. The cross-linker is consumedlightly in cross-linking the collagen. A HA solution is added as alubricant and cross-linker scavenger. The final formulation has a coreof cross-linked HA with collagen lightly cross-linked and surroundingthe HA core. The additional soluble HA consumes the residualcross-linker and adds a lubrication benefit. Generally, any cross-linkerthat induces covalent bonds linking HA and collagen molecules may beused. Examples of such cross-linkers include but are not limited to VS,BDDE, ultraviolet radiation, gamma radiation, electron beam radiation,and glutaraldehyde.

Cross-linking of HA and collagen with VS is provided here as an example.Base is added to HA (30 mg/ml or higher) to raise the pH to 9-12. VS(500-10,000 ppm, and preferably 5000 ppm) is added. Cross-linking of HAby VS is allowed to proceed at an elevated temperature from 50° C. to60° C. for about 1 hour. The cross-linked HA is cooled and acidiccollagen (10-50 mg/ml, and preferably 30 mg/ml; pH 2-3) is added tobring the pH to ˜7 (e.g., 6-8). The reaction is allowed to proceedovernight at room temperature. Soluble HA (3-15% weight/volume, andpreferably 5-10% weight/volume) is added. Optionally, lidocaine can beadded for a final lidocaine concentration of 2.7-3.3 mg/ml. The reactionis allowed to proceed for 8 hours-2 weeks at room temperature and pH7.0-7.6. The scavenger HA not only decreases the amount of the residualVS but also functions as a coating agent which in turn reduces theextrusion force of the resulting implant (HAVSCgn). The HAVSCgn containslow residual VS (10 ppm or less) and has low extrusion force (e.g., 50Newtons or less) through a fine gauge needle (>24 gauge). 5-50% aminegroups on the collagen are modified by VS (i.e., loss of free aminegroups) in the implant.

The HAVSCgn is a formulation with a high elastic modulus (>200 Pascals)yet low extrusion plateau. The elastic modulus (G′) may be determinedwith a Bohlin Rheometer by measuring the ratio of the stress to thestrain multiplied by the cosine of the phase angle. The extrusion forcemay be determined as follows: The implant is filled into a 1 ccUniversal Syringe. A needle (e.g., 30 G½ needle, Becton DickinsonPrecision Glide) is then fit into the syringe and luer locked. Thesyringe filled with the implant and equipped with the needle is placedinto the grip of Instron, Model 4201, and the plunger is displaced at aconstant speed of 50 mm/minute. The force plateau is then calculated andreported in Newtons.

A cross-linked HA-collagen implant of the invention can be furtherhomogenized and screened by forcing the formulation through a screen ofdefined pore size. The formulation can also be filled into syringesfitted with a #25 or larger gauge needle for injection. In the case offormulations used for dermal augmentation, the term “injectable” meansthat the formulation can be dispensed from syringes having a gauge aslow as #25 under normal manual pressure with a smooth extrusion plateau.

The steps described above for preparing cross-linked HA-collagen andfilling the implant into syringes are preferably carried out in sterileconditions using sterile materials.

The invention provides a soft tissue augmentation injectable that mimicsthe natural components of the skin. The composition of the presentinvention may be injected intradermally or subcutaneously to augmentsoft tissue and to repair or correct congenital anomalies, acquireddefects, or cosmetic defects. Examples of such conditions includecongenital anomalies such as hemifacial microsomia, malar and zygomatichypoplasia, unilateral mammary hypoplasia, pectus excavatum, pectoralisagenesis (Poland's anomaly), and velopharyngeal incompetence secondaryto cleft palate repair or submucous cleft palate (as a retropharyngealimplant); acquired defects (post traumatic, post surgical, or postinfectious) such as depressed scars, subcutaneous atrophy (e.g.,secondary to discoid lupus erythematosus), keratotic lesions,enophthalmos in the enucleated eye (also superior sulcus syndrome), acnepitting of the face, linear scleroderma with subcutaneous atrophy,saddle-nose deformity, Romberg's disease, and unilateral vocal cordparalysis; and cosmetic defects such as glabellar frown lines, deepnasolabial creases, circum-oral geographical wrinkles, sunken cheeks,and mammary hypoplasia.

The present invention provides a HA-collagen filler for augmenting andfilling soft tissue defects and voids with a material that plumps andbulks the soft tissue. The cross-linked HA-collagen of the invention isparticularly useful for deep dermal correction and sculpting. Thesuperior composition and persistence makes it ideal for areas that arehard to correct and where a biocompatible bolus can provide mechanicalstrength to the body.

The following example is intended to illustrate, but not to limit, thescope of the invention. While such example is typical of those thatmight be used, other procedures known to those skilled in the art mayalternatively be utilized. Indeed, those of ordinary skill in the artcan readily envision and produce further embodiments, based on theteachings herein, without undue experimentation.

EXAMPLE Preparation of HAVSCgn

HA was obtained from Lifecore Biomedical, Chaska, Minn. Base (NaOH) wasadded to HA to adjust the pH to a range of 9-12. Chemically pure VS (TCIAMERICA, 9211 N. Harborgate Street, Portland, Oreg. 97203, U.S.A) wasthen added to HA to allow the cross-linking reaction to proceed at anelevated temperature between 50° C. and 60° C. The cross-linked HA wascooled and collagen added.

Purified, type I, pepsin-digested human collagen was obtained fromInamed Biomaterials, 48490 Milmont Drive, Fremont, Calif. 94538. It wasprecipitated by raising the pH to 7.0-7.6 with a sodium phosphatesolution and then centrifuging at 17000×g for 5-7 minutes. Thesupernatant was aseptically decanted from the centrifuge bottle. Thecollagen pellet was aseptically suctioned into a homogenization vesseland homogenized. 0.05 M HCl buffer and sterile filtered WFI (water forinjection) were mixed with the homogenate to lower the final pH to 2-3.

The collagen was added to the cross-linked HA while the collagen wassoluble. The VS was consumed and as a result the collagen was lightlycross-linked to collagen and HA. Additional HA in a solution was addedat the end to consume the residual VS and to add a lubrication benefit.The final formulation had a core of cross-linked HA with collagenlightly cross-linked to the HA core. The additional HA was cross-linkedto collagen and HA to enhance the formulation. The residual VS afteraddition of the free HA solution, as detected by reversed-phase HPLC, isindicated in Table 1.

TABLE 1 Consumption of Divinyl Sulfone Formulation Residual DivinylSulfone 30 mg/ml HA + 5000 ppm VS 1776 ppm 30 g/ml HA + 5000 ppm VS + 18 ppm 5% HA solution 30 mg/ml HA + 5000 ppm VS +   0 ppm 10% HAsolution

VS primarily reacts with the amine groups on collagen and the hydroxylgroups on HA. The percentage of modified amine groups on collagen wasdetermined by a free amine assay using TNBS (trinitrobenzene sulfonate).Unmodified collagen was used as a control. The HAVSCgn, 2674-1,formulation had about 15% of the amine groups modified.

The reaction of VS with HA proceeds predominately with the hydroxylgroups of HA and the vinyl groups of VS and results in the formation ofan ether bond.

R—OH+CH2=CH—SO2−CH═CH2→R—O—CH2−CH2—SO2−CH2−CH2−O—R

FIG. 4 shows FT-IR spectra obtained for uncross-linked HACgn (a mixtureof uncross-linked HA and uncross-linked collagen) and the HAVSCgnformulation. The representative absorption peaks in both samples wereassigned to primary amines at 3500-3300 cm-¹ and alcohol or OH groups at3650-3200 cm-¹ which overlaps the ether bond at 1300 cm-¹. Cross-linkedHAVSCgn had an ether peak whereas the uncross-linked sample did not havea peak at 1300 cm-¹.

Rheology of HAVSCgn

FIG. 2 shows a frequency sweep test using a Bohlin Rheometer todetermine the elastic modulus (G′) and the viscous modulus (G″) ofHAVSCgn. The elastic modulus reflects the elasticity of a material, andindicates the strength and stability of the HAVSCgn three-dimensionalstructure. Stability of the three-dimensional structure refers to theability of HAVSCgn to resist deformation to maintain a three-dimensionalform. The higher the value of G′ at a given frequency, the more elasticthe HAVSCgn will be. The viscous modulus reflects the viscosity of amaterial. The higher the value of G″ at a given frequency, the moreviscous the HAVSCgn gel composition will be.

Enzyme Resistance

Hyaluronidase effect on elastic modulus was studied for HAVSCgn andCaptique™ (a formulation of HA cross-linked with VS). Hyaluronidase wasobtained from Worthington Biochemical Corporation (730 Vassar Avenue,Lakewood, N.J. 08701; catalog number L5002592). Implants were weighed toabout 2±0.1 gram. 5 mg hyaluronidase was diluted with 10 ml PBS solutionto ˜50 U/ml. 0.020 ml hyaluronidase solution was added to each 2 gramimplant sample. The samples were incubated at 37° C. for 30 minutes, 1.5hours, 3 hours, and 5 hours. The elastic modulus and phase angle weretested at 5 Hz using a Bohlin Rheometer CVO-100 at 20° C. with a gap of500 mm and a parallel plate (PP20). As shown in FIG. 3, HAVSCgn was moreresistant to hyaluronidase than Captique™.

Biocompatibility

The biocompatibility of HAVSCgn was tested and demonstrated to bebiocompatible. The safety of the HAVSCgn was assessed through acytotoxicity study and multiple rabbit subcutaneous implantationstudies. The cytotoxicity study was performed using the ISO ElutionMethod. As shown in Table 2, HAVSCgn caused no cell lysis or toxicity.

TABLE 2 Cytotoxicity of HAVSCgn Test Score Confluent monolayer 0 Percentrounding 0 Percent cells without intracytoplasmic granules 0 Percentlysis 0 Grade 0 Reactivity None

A rat subcutaneous implantation study was performed to determine thepersistence of HAVSCgn. As part of the rat implantation study, amacroscopic evaluation of the implant site was performed. There was nocapsule formation or adverse reaction for all time points studied (Table3).

Effectiveness

TABLE 3 Rat Subcutaneous Gross and Microscopic Evaluations Time PointHAVSCgn  4 weeks No capsule formation or adverse reaction. Non irritant. 9 weeks No capsule formation or adverse reaction. Non irritant. 13weeks No capsule formation or adverse reaction. Non irritant. 24 weeksNo capsule formation or adverse reaction. Non irritant.

To assess effectiveness, the persistence of HAVSCgn (2674-1) relative toCosmoPlast® and Captique™ was evaluated using wet weight recovery in therat subcutaneous model which is considered to be a good measure of animplant's ability to maintain wrinkle correction (McPherson et al.,1988, Development and Biochemical Characterization of InjectableCollagen, J Dermatol Surg Oncol 14, Suppl 1). As shown in FIG. 5,HAVSCgn had greater wet weight recovery than CosmoPlast® and Captique™after implantation.

All patents and articles cited herein are incorporated by reference intheir entirety.

What is claimed is:
 1. A process for preparing an injectable compositionuseful for correcting mammary hypoplasia, the process comprising: (1)contacting hyaluronic acid (HA) with a cross-linker to allowcross-linking of the HA by the cross-linker, thereby forming a firstcomposition comprising a crosslinked HA; (2) contacting the crosslinkedHA with collagen to form a second composition comprising a collagenlayer crosslinked to and surrounding a crosslinked HA core; (3)homogenizing the second composition to form an injectable composition,the composition being injectable under normal manual pressure though a25 gauge needle, thereby preparing the injectable composition useful forcorrecting the mammary hypoplasia.
 2. The process of claim 1, whereinthe cross-linker is 1,4-butanediol diglycidyl ether (BDDE).
 3. Theprocess of claim 1, wherein the concentration of the HA for forming thefirst composition is about 30 mg/ml or higher.
 4. The process of claim1, wherein the pH of the HA for forming the first composition is about9-12.
 5. The process of claim 1, wherein the collagen for contacting thefirst composition is soluble.
 6. The process of claim 1, wherein theconcentration of the collagen for contacting the first composition isabout 10-50 mg/ml.
 7. The process of claim 1, wherein the concentrationof the collagen for contacting the first composition is about 30 mg/ml.8. The process of claim 1, wherein the pH of the collagen for contactingthe first composition is about 2-3.
 9. The process of claim 1, whereinthe pH of the second composition is about 6-8.
 10. The process of claim1, wherein the pH of the second composition is about
 7. 11. The processof claim 1, further comprising adding a local anesthetic agent to thesecond composition.
 12. The process of claim 11, wherein the localanesthetic agent is lidocaine.
 13. The process of claim 1 furthercomprising the step of adding soluble HA to the second composition. 14.An injectable composition useful for correcting mammary hypoplasia in apatient, the composition made by the process of: (1) contactinghyaluronic acid (HA) with a cross-linker to allow cross-linking of theHA by the cross-linker, thereby forming a first composition comprising acrosslinked HA; (2) contacting the crosslinked HA with collagen to forma second composition comprising a collagen layer crosslinked to andsurrounding a crosslinked HA core; and (3) homogenizing the secondcomposition to form an injectable composition, thereby preparing theinjectable composition useful for correcting the mammary hypoplasia. 15.The composition of claim 14, wherein the cross-linker is 1,4-butanedioldiglycidyl ether (BDDE).
 16. The composition of claim 14, wherein theconcentration of the HA for forming the first composition is about 30mg/ml or higher.
 17. The composition of claim 14, wherein the pH of theHA for forming the first composition is about 9-12.
 18. The compositionof claim 14, wherein the collagen for contacting the first compositionis soluble.
 19. The composition of claim 14, wherein the concentrationof the collagen for contacting the first composition is about 10-50mg/ml.
 20. The composition of claim 14, wherein the concentration of thecollagen for contacting the first composition is about 30 mg/ml.
 21. Thecomposition of claim 14, wherein the process further comprises addinglidocaine to the second composition.
 22. A method for volumizing ahypoplastic breast in a patient comprising introducing the compositionof claim 14 into the breast of a patient.